Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo

Cytotoxic activity of the histone deacetylase inhibitorpanobinostat (LBH589) in anaplastic thyroid cancerin vitro and in vivo

Maria Graziella Catalano1, Mariateresa Pugliese1, Eleonora Gargantini1, Cristina Grange2, Benedetta Bussolati2,

Sofia Asioli3, Ornella Bosco1, Roberta Poli1, Alessandra Compagnone4, Andrea Bandino4, Franco Mainini5,

Nicoletta Fortunati6 and Giuseppe Boccuzzi1,6

1 Department of Clinical Pathophysiology, University of Turin, Torino, Italy2 Department of Internal Medicine, University of Turin, Torino, Italy3 Department of Biomedical Sciences and Human Oncology, University of Turin, Torino, Italy4 Department of Experimental Medicine and Oncology, Biochemistry Section, University of Turin, Torino, Italy5 Novartis Farma S.p.A., Origgio (VA), Italy6 Oncological Endocrinology, AUO San Giovanni Battista, Torino, Italy

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal clinical course, being resistant to multimodal treatments. Microtubules,

a/b tubulin heterodimers, are crucial in cell signaling, division and mitosis and are among the most successful targets for

anticancer therapy. Panobinostat (LBH589) is a potent deacetylase inhibitor acting both on histones and nonhistonic proteins,

including a-tubulin. In vitro LBH589, evaluated in three ATC cell lines (BHT-101, CAL-62 and 8305C), resulted in impairment of

cell viability, inhibition of colony formation, cell cycle arrest and apoptosis induction. Mechanistically, we showed that

LBH589 not only affected the expression of p21 and cyclin D1, but markedly determined microtubule stabilization as

evidenced by tubulin acetylation and increased tubulin polymerization. In a SCID xenograft model implanted with CAL-62 cells,

the cytotoxic properties of LBH589 were confirmed. The drug at the dose of 20 mg/kg significantly impaired tumor growth

(final tumor volume 2.5-fold smaller than in untreated animals); at this dose, no relevant side effects were observed. In

tumors of treated animals, a significant reduction of Ki67, which was negatively correlated with tubulin acetylation, was

observed. Moreover, acetyl-tubulin levels negatively correlated with tumor volume at sacrifice, reinforcing the opinion that

tubulin acetylation has a role in the inhibition of tumor growth. In conclusion, LBH589, acting on both histones and

nonhistonic proteins in anaplastic thyroid cancer, appears to be a promising therapeutic agent for the treatment of this kind

of cancer which is known not to respond to conventional therapy.

Anaplastic thyroid carcinoma (ATC), which accounts for 1%of thyroid tumors, is one of the most lethal malignancies,with a rapidly fatal clinical course.1 Management of ATC hasnot yet been standardized; multimodal treatments including

surgical resection associated with radiotherapy and combina-tion chemotherapy are currently used2 but efforts to improvesurvival have been disappointing.3

Deacetylase inhibitors (DCIs) represent a class of thera-peutic agents with broad activity toward cancer cells, namelyactivation of pro-apoptotic pathways and inhibition of anti-apoptotic ones, induction of cell differentiation, antiangio-genic activity and synergism with established and experimen-tal cancer therapeutics.4–10 This spectrum of activity cannotbe explained only by transcription modulation; in fact, sev-eral substrates different to histones such as p53, Hsp90 anda-tubulin11 have been identified. To date, some DCIs havedemonstrated anti-tumor activity against undifferentiatedthyroid carcinomas; however, they have shown a low po-tency, as they are effective only at millimolar or micromolarconcentrations.6,12 We already showed that the only clinicallyavailable DCI valproic acid (VPA) has cytotoxic and pro-differentiating effects on poorly differentiated thyroid cancercells,6,7 but it is poorly effective against ATC if used alone;nevertheless, in ATC cells, VPA efficiently potentiates theeffects of doxorubicin and paclitaxel.8,9

Key words: anaplastic thyroid cancer, panobinostat, histone

deacetylase inhibitors

Additional Supporting Information may be found in the online

version of this article

Conflict of interest: F.M. is an employee of Novartis Farma SpA,

Italia

Grant sponsors: Fondazione CRT, Turin (Project ‘‘Alfieri 2007’’),

MIUR, Regione Piemonte

DOI: 10.1002/ijc.26057

History: Received 25 Oct 2010; Accepted 14 Feb 2011; Online 11

Mar 2011

Correspondence to: Giuseppe Boccuzzi, Dipartimento di

Fisiopatologia Clinica, Via Genova 3, 10126 Torino, Italy, Tel.:

þ39-011-670-5399, Fax: þ39-011-670-5366, E-mail: giuseppe.

[email protected]

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Int. J. Cancer: 130, 694–704 (2012) VC 2011 UICC

International Journal of Cancer

IJC

Panobinostat (LBH589) is a potent DCI that belongsto the cinnamic hydroxamic acid class of compounds.13 Itinhibits HDAC1, 3–6 at nanomolar concentrations; it is cur-rently in Phase I clinical evaluation in advanced refractorysolid tumors and hematological malignancies,14–16 but nodata are available about the use of this DCI in ATC.

In this work, we show for the first time in vitro and in vivothat LBH589 has cytotoxic effects on ATC, a disease whichdoes not respond to any standard or experimental therapy.

Material and MethodsCell lines and reagents

The study was performed on three different anaplasticthyroid cancer cell lines: BHT-101, CAL-62 and 8305C. Cellswere purchased from Deutche Sammlung von Mikroorganis-men and Zellculturen (Braunschweig, Germany), which certi-fies the origin and identity of the cells; moreover, BHT-101and CAL-62 cells are included in the list of recently authenti-cated unique thyroid cancer cell lines.17

LBH589 was provided by Novartis Pharma AG (Basel,Switzerland), prepared as a 5 mM stock solution in DMSOand stored at �80�C.

Cytotoxic activity

Cells were seeded at 1 � 103 cells/well in 96-well plates(Corning, New York, NY) in culture medium plus 10% heat-inactivated FCS (Sigma, St. Louis, MO). After 24 hr, cellswere exposed to increasing concentration of LBH589 (5–100nM). At 24, 48 and 72 hr incubation, cell viability wasassessed using the Cell Proliferation Reagent WST-1 (RocheApplied Science, Penzberg, Germany), following the manu-facturer’s instructions. This is a colorimetric assay for thequantification of cell viability and proliferation, based oncleavage of the tetrazolium salt WST-1 by mitochondrialdehydrogenases in viable cells. Briefly, 10 ll of WST-1 wereadded to each well. After 1-hr incubation, absorbance at 450nm was measured using a plate reader (Model 680 Micro-plate Reader, Bio-Rad, Hercules, CA). Four replicate wellswere used to determine each data point.

Three response parameters, median growth inhibition(GI50), total growth inhibition (TGI) and median lethal con-centration (LC50) were calculated for each cell line. The GI50corresponds to the concentration of the compound thatinhibits 50% net cell growth; TGI value is the concentrationof the compound leading to TGI; LC50 value is the concen-tration of the compound leading to 50% net cell death.

The higher dose of LBH589 used in our study (100 nM)is within the range of the clinically attainable plasma concen-trations as they result from a Phase I study in patients withrefractory hematological malignancies.14

Colony forming assay

Cells were seeded (100 cells/well) into 24-well plates and leftovernight to attach. After 24 hr, the cells were treated withincreasing concentrations of LBH589 (5–100 nM) for 72 hr.

Cells were then cultured for 10 days and subsequently fixedin methanol and stained with crystal violet. The colonieswere then photographed with Kodak 1D Image equipment.

Apoptosis detection

Cell death detection ELISA. For apoptosis studies, 1 � 103

cells were seeded in 96-well plates and treated with LBH589,using the same schedule as for the viability assay. After treat-ment, apoptosis was evaluated using Cell Death DetectionELISAPLUS (Roche Applied Science, Penzberg, Germany) fol-lowing the manufacturer’s instructions. This assay is basedon a quantitative sandwich-enzyme-immunoassay-principleusing monoclonal antibodies directed against DNA and his-tones, respectively. The assay provides the specific determina-tion of mono- and oligo-nucleosomes in the cytoplasm frac-tion of cell lysates. Apoptosis was expressed as enrichmentfactor, calculated as a fraction of the absorbance of treatedcells versus untreated controls.

Caspase 3 activity assay. 1 � 106 cells were seeded in 75cm2 flasks and exposed to LBH589 as above. After drugtreatments, caspase 3 was determined using a colorimetricassay kit (R&D Systems, Minneapolis, MN) and following themanufacturer’s instructions. Briefly, cells were lysed and incu-bated with the colorimetric substrate DEVD-pNA for 2 hr at37�C. After incubation, the chromophore was quantifiedspectrophotometrically at 405 nm.

Cell cycle analysis

Cells were treated with LBH589 up to 72 hr. At differenttimes (24, 48 and 72 hr), all cells were collected, fixed in 70%ethanol for 30 min on ice and incubated in propidium iodidesolution (20 lg/ml propidium iodide, 0.2 mg/ml RNAseA inPBS) for 1 hr at room temperature. The cell population wasanalyzed by flow cytometry.

Gene expression

Cells (1 � 106) were seeded in 75 cm2 flasks and treated asdescribed above. After 24 hr treatment, total RNA wasextracted using TRIzol Reagent (Invitrogen, Paisley, UK).DNase I was added to remove remaining genomic DNA.

A total of 1 lg of total RNA was reverse-transcribed withiScript cDNA Synthesis Kit (BioRad Laboratories), followingthe manufacturer’s protocol.

Primers (Supporting Information Table S1) were designedusing Beacon Designer 5.0 software according to parametersoutlined in the BioRad iCycler Manual. Specificity of primerswas confirmed by BLAST analysis. Real-time PCR was per-formed using a BioRad iQ iCycler Detection System (BioRadLaboratories) with SYBR green fluorophore. Reactions wereperformed in a total volume of 25 ll-included 12.5 ll IQSYBR Green Supermix (BioRad Laboratories), 1 ll of eachprimer at 10 lM concentration and 5 ll of the previouslyreverse-transcribed cDNA template. Protocol for primer setwas optimised using seven serial 5� dilutions of template

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cDNA obtained from cells in basal conditions. The protocolused is as follows: denaturation (95�C for 5 min), amplificationrepeated 40 times (95�C for 15 sec, 60�C for 30 sec). A melt-ing curve analysis was performed following every run to ensurea single amplified product for every reaction. All reactionswere carried out at least in triplicate for each sample. Resultswere normalized using the geometric mean for three differenthousekeeping genes (b-actin, b2-microglobulin and L13A) andexpressed as relative expression fold versus untreated controls.

Immunoblotting

After treatment with LBH589 (5–100 nM), BHT-101 andCAL-62 cells were harvested and lysed in the presence of 200ll RIPA buffer (1% NP40, 0.5% desoxycholate sodium, 0.1%SDS in PBS pH 7.4, with 10 mg/ml PMSF, 30 ll/ml aprotininand 100 mM sodium orthovanadate) and incubated on icefor 30 min; cells were then centrifuged for 20 min at 15,000gat 4�C, and clear supernatants used. SDS-PAGE was per-formed on gels, loading 40 lg protein/well. Separated pro-teins were electro-transferred onto PVDF membrane andprobed with the following antibodies: anti-acetyl-histone H3(raised against a KLH conjugate peptide corresponding to theamino acids 1–20 of histone H3; 1:4,000 dilution, 06-599,Millipore, Billerica, MA); anti-histone H3 (sc-10809, 1:500dilution, Santa Cruz Biotechnology, Santa Cruz, CA); anti-acetyl-histone H4 (raised against a KLH conjugate peptidecorresponding to the amino acids 2–19 of histone H4; 06-598, 1:1,000 dilution, Upstate, Lake Placid, NY); anti-histoneH4 (sc-10810, 1:500 dilution, Santa Cruz Biotechnology,Santa Cruz, CA); anti-acetyl-a-tubulin (clone 6-11B-1,1:8,000 dilution, Sigma, St. Louis, MO); anti-a-tubulin (clone6-11B-1, 1:2,000 dilution, Sigma, St. Louis, MO); anti-p21(sc-397, 1:200 dilution, Santa Cruz Biotechnology, SantaCruz, CA) and anti-cyclin D1 (sc-718, 1:400 dilution, SantaCruz Biotechnology, Santa Cruz, CA). The membrane wasthen stripped and reprobed with an anti-GAPDH antibody(1:10,000 dilution, Sigma, St. Louis, MO) to check proteinloading. Proteins were detected with Pierce Super Signalchemiluminescent substrate following the manufacturer’sinstructions. Bands were photographed and analyzed usingKodak 1D Image Analysis software.

Immunofluorescence microscopy

Cells (3 � 105) were seeded in 6-cm dishes. After 24 hr, cellswere treated with LBH589 (5–100 nM). Then cells were fixedin acetone/methanol (1:1) at �20�C for 20 min, washed withPBS containing 0.5% Triton X-100, 0.05% NaN3 and 1%Horse Serum and incubated with anti-acetyl-a-tubulin (clone6-11B-1, 1:8,000 dilution, Sigma, St. Louis, MO) or anti-a-tubulin (clone 6-11B-1, 1:2,000 dilution, Sigma, St. Louis,MO) antibodies in PBS at 4�C overnight. Then, cells werewashed with PBS containing 0.5% Triton and 0.05% NaN3

for 10 min for three times followed by detection with anti-mouse Cy3-conjugated secondary antibody (1:1,000), (GEHealthcare Europe, GmbH, Milan, Italy) in PBS plus 0.5%

Triton and 0.05% NaN3 for 2 hr. Nuclear staining wasobtained by treating cells with Hoechst 33258 (500 ng/ml inDMSO) in PBS. Cells were washed twice with distilled waterand mounted with 50% glycerol-PBS media.

Tubulin polymerization assay

BHT-101 and CAL-62 cells were treated with LBH589 (5–100nM). After 24 hr treatment, quantitative drug-induced tubulinpolymerization was done as previously described18 and ali-quots of each sample were analyzed by SDS-PAGE. Immuno-blottings were done using monoclonal anti-a-tubulin antibody(1:2,000 dilution, Sigma, St. Louis, MO). The percent of poly-merized tubulin (P) was calculated over the total of tubulin(T) times 100 [P/T] based on densitometric analysis.

Xenograft studies

Animal experiments were approved by the ethical committeeof the University of Turin. Suspension of 2 � 106 CAL-62cells were injected under the skin in the flank of 20 7-week-old female SCID mice (Charles River Laboratories, Italy). AsLBH589 had the higher LC50 on CAL-62 cells (SupportingInformation Table S2), this cell line was used for the xeno-graft studies. When tumors reached a minimum volume of100 mm3, animals were randomly assigned to different treat-ment groups: not treated (controls, n ¼ 5), 10 mg/kg (n ¼5), 20 mg/kg (n ¼ 5) and 30 mg/kg (n ¼ 5) LBH589. Intra-peritoneal injection of LBH589 or vehicle lasted 21 days(5 days/week). Tumor volume and mice body weight wereassessed every 7 days. Tumors were measured with calipersand volumes were calculated with the formula a2 � b � 0.5,where a is the shortest diameter and b is the diameter per-pendicular to a. When treatment ended, pieces of tumorwere either fixed in 10% buffered formalin, or put in AllPro-tect Tissue Reagent (Life Science, Italy). Blood and organsincluding liver, kidney, lung, spleen and intestine were col-lected for treatment side effect evaluation. Total proteinsfrom tumors were extracted and used for immublottingdetection of acetyl-histone and acetyl-tubulin as above.

Histology

Paraffin sections (4 lm) were cut for H&E staining andimmunohistochemistry. Ki67 positive cells were heterogene-ously distributed throughout the tumor. The Ki67 labelednuclei was evaluated in the tumor areas where these markerswere predominant (hot spots). Given that cell counting onnumerous microscopic fields is known to be a source ofinter- and intra-observer variability, a digital camera (Olym-pus Q-colour 3) with area-based image analysis software(Dot-Slide 1.2 version) was used. Immunostained sectionswere screened under the microscope, and the areas contain-ing the greatest number of Ki67 stained nuclei were outlinedwith a slide-pointer, allowing easy localization during imageanalysis. The Ki67 was calculated as the ratio between thelabeled and the total nuclear areas. Only nuclei with astrongly positive label were counted. The ten fields with the

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highest density of positive nuclei were captured. A mean of3,000 tumor cells per case (range, 2,000–3,800) was counted.

TUNEL staining

Apoptosis was evaluated on slices of tumors recovered at theend of the in vivo experiment in SCID mice by terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay (ApopTag Plus, Millipore, Billerica, MA);paraffin embedded slices of tumors were deparaffined andTUNEL assay were performed following the manufacturer’sinstruction. After excluding areas of necrosis, we counted thenumber of apoptotic cells per field in 20 randomly chosensections, using 400� magnification.

Statistical analysis

Data are expressed throughout as means 6 SD, calculatedfrom at least three different experiments. Comparison

between groups was performed with analysis of variance(one-way ANOVA) and the threshold of significance wascalculated with the Bonferroni test. Statistical significance wasset at p < 0.05.

ResultsHistone and a-tubulin acetylation

LBH589 caused hyper-acetylation of both histones H3 andH4 and tubulin (Fig. 1, panel a), confirming that all the celllines used in the study were good models to study the effectsof this DCI.

Cytotoxic activity

LBH589 exerted cytotoxic activity in ATC cells as demon-strated by impairment of cell viability (Fig. 1 panel b andSupporting Information Table S2) and inhibition of colonyformation (Fig. 1, panel c). Cytotoxic activity was explained

Figure 1. LBH589 effect on histone and tubulin acetylation (panel a). Acetylation of histones H3 and H4 and tubulin in BHT-101, CAL-62

and 8305C cells incubated for 24 hr with LBH589 (5–100 nM) was assessed by Western blotting. Equal loading and transfer were verified

by reprobing the membranes with anti-H3, anti-H4 and anti-tubulin antibodies, respectively. The figure shows a typical experiment. LBH589

effect on cell viability (panel b). Cells were incubated with LBH589 (5–100 nM). At 0, 48 and 72 hr after treatment, cell viability was

determined by the WST-1 method, and expressed as the ratio between treated cells and untreated controls. LBH589 effect on colony

formation (panel c). Cells were seeded in 24-well plates and treated with LBH589 for 72 hr. Cells were cultured for 10 days and

subsequently stained with crystal violet.

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by both apoptosis induction and cell cycle arrest. LBH589induced apoptosis in a dose-dependent manner, as demon-strated by quantification of nucleosome formation (Fig. 2,panel a) and confirmed by both caspase-3 activity (Support-ing Information Fig. S1) and flow cytometry, revealing anincrease of the sub-G1 population of LBH589-treated cellsafter 72 hr treatment (Fig. 2, panels b and c). In addition toapoptosis induction, LBH589 caused G1 cell cycle arrest atlow concentrations, e.g., between 5 and 25 nM; higher doses(25–100 nM) resulted in G2-M arrest (Table 1). The effectwas already evident after 24 hr of treatment. The presence ofmultiple nuclei (Supporting Information Fig. S2) confirmsthat LBH589 causes mitotic arrest. In the three different ana-plastic cancer cell lines, the cytotoxic effect of LBH589 wassimilar, even though the drug showed the lower GI50 onBHT-101 cells (Supporting Information Table S2).

LBH589 effect on cell cycle- and apoptosis-related

gene expression

To further clarify the mechanisms behind LBH589-inducedcell cycle arrest and apoptosis induction, we examined the

effects of the drug on the expression of some of the genesinvolved in cell cycle and apoptosis control. Low concentra-tions of LBH589 affected mRNA expression of p21 andcyclin D1 (Fig. 3, panels a and b). At protein level, weobserved the upregulation of p21 starting from the concen-tration of 50 nM, whereas the downregulation of cyclin D1was evident from 50 nM in BHT-101 and at 100 nM inCAL-62 cells (Fig. 3, panel c). The modulation of gene tran-scription was furthermore supported by the induction ofacetylation of histones H3 and H4 (Fig. 1, panel a).

As far as apoptosis regulating genes are concerned,regardless of the presence of G2 arrest and apoptosis induc-tion, no modulations of the expression of the anti-apoptoticgenes (e.g., Bcl-2 and Bcl-xl) as well as of the pro-apoptoticgenes (e.g., Bax and Bak) were observed (data not shown).

LBH589 effect on microtubule structure

Because tubulin deacetylation is associated with microtubuledepolymerization, accumulation of acetylated tubulin follow-ing LBH589 treatment of thyroid cancer cells should leadto microtubule stabilization. Using immunofluorescence, we

Figure 2. ELISA detection of DNA-histone complex (panel a). Cells were incubated for 24 hr with LBH589 (5–100 nM). The enrichment

factor was calculated as the ratio between the absorbance measurement of treated cells and untreated controls. Results are expressed

as mean 6 SD; n ¼ 3. Significance vs. untreated cells (0): *p < 0.05; **p < 0.01; ***p < 0.001. LBH589 effect on cell cycle (panel b).

The analysis was performed by flow cytometry after 72 hr treatment with LBH589 (5–100 nM). Sub-G1 accumulation after treatment

with LBH589 (panel c). Results are expressed as means 6 SD; n ¼ 3. Significance vs. untreated cells (0): *p < 0.05; **p < 0.01;

***p < 0.001.

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Table 1. Cell cycle progression of viable BHT-101, CAL-62 and 8305C cells after LBH589 treatment

LBH589 (nM)

0 5 10 25 50 100

BHT-101 G1 60.8 6 1.0 84.6 6 1.8*** 86.3 6 1.4*** 76.1 6 3.1** 67.9 6 2.8 61.6 6 3.7

S 32.4 6 1.2 11.2 6 1.7††† 7.7 6 0.7††† 9.5 6 1.6††† 11.4 6 2.4†† 16.7 6 4.3†

G2/M 6.9 6 1.7 4.2 6 1.0 5.9 6 1.1 14.4 6 2.2‡ 20.6 6 2.6‡‡ 21.6 6 3.7‡‡

CAL-62 G1 41.3 6 1.7 52.4 6 5.1* 56.5 6 2.1** 52.6 6 4.4* 43.4 6 6.1 40.9 6 5.4

S 47.0 6 3.1 35.6 6 6.4 25.1 6 5.7† 17.8 6 3.1†† 15.3 6 2.1†† 17.2 6 3.6††

G2/M 10.1 6 1.2 16.2 6 2.2 19.2 6 3.9 37.7 6 4.8‡‡ 43.3 6 7.5‡‡ 43.8 6 5.7‡‡

8305C G1 50.1 6 2.1 69.7 6 2.9* 69.0 6 2.2* 51.6 6 3.3 39.4 6 2.4 42.1 6 5.4

S 24.0 6 2.0 17.7 6 2.5 14.1 6 1.3† 9.7 6 0.2†† 10.5 6 1.2†† 10.3 6 2.0††

G2/M 16.8 6 9.3 17.8 6 3.9 21.4 6 3.0 38.6 6 3.3‡‡ 50.0 6 2.0‡‡ 47.5 6 2.2‡‡

G1 significance vs. untreated cells: *p < 0.05; **p < 0.01; ***p < 0.001. S significance vs. untreated cells: †p < 0.05; ††p < 0.01; †††p < 0.001.G2-M significance vs. untreated cells: ‡p < 0.05; ‡‡p < 0.01.

Figure 3. LBH589 effect on the mRNA expression of p21 (panel a) and cyclin D1 (panel b). mRNA was evaluated with Real Time PCR.

Results are normalized for three different housekeeping genes (b-actin, b2-microglobulin and L13A) and expressed as relative expression

fold vs. untreated controls (0). Protein expression of p21 and cyclin D1 (panel c) analyzed by Western blot. Equal loading and transfer were

verified by reprobing the membranes with GAPDH antibody.

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observed that altered microtubule structure accompaniesthe increase in tubulin acetylation. As shown in Figure 4,panel a, in untreated cells, microtubules were seen as fine,diffuse and evenly distributed cytoplasmatic networks. Incontrast, at the dose of 25 nM, tumor cells showed relativelythicker microtubule bundles organized in parallel lines, espe-cially at the periphery of the nuclei. To further probe theeffect of LBH589 treatment on microtubule stabilization, acell-based tubulin polymerization assay was done in both

BHT-101 and CAL-62 cells. The assay is based on the factthat stabilized microtubules remain insoluble when extractedin a hypotonic buffer and, therefore, remain in the pelletafter centrifugation. Conversely, the soluble tubulindimers remain in the supernatant. As shown in Figure 4,panels b and c, LBH589 resulted in a dose-dependentincrease in polymerized tubulin in both BHT-101 and CAL-62 cells, without any changes in total tubulin, as compared tountreated cells.

Figure 4. LBH589 effect on microtubule structure and acetylation (panel a). Cells were treated with 25 nM LBH589 and microtubules

were visualized by immunofluorescence labeling using antibodies against tubulin and acetylated tubulin. LBH589 effect on tubulin

polymerization (panel b). Western blot analysis against the polymerized (P), soluble (S) and total (T) fractions of tubulin in protein lysates

from BHT-101 and CAL-62 cells treated for 24 hr with LBH589 (5–100 nM). % polymerized tubulin: relative percentage of polymerized

vs. total tubulin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 5. LBH589 effect on tumor volume (panel a). Tumor volume was assessed at time 0 and at days 7, 14 and 21 of treatment. LBH589

effects on tubulin acetylation (panel b). Acetylation of tubulin was evaluated by Western blotting in tumor lysates and expressed as ratio in

respect with total tubulin. Significance vs. controls: *p < 0.05; **p < 0.01. LBH589 effect on ki67 (panel c). A representative picture for

Ki67 staining is presented and results are presented in box plots. Significance vs. controls: *p < 0.05. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

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In vivo LBH589 effects

The anti-tumor effect of LBH589 was assessed in vivo in aSCID mice model implanted with CAL-62 cells. As shown inFigure 5 panel a, treatment with 20 mg/kg LBH589 resultedin a significant reduction of tumor growth with respect tocontrols.

In mice treated with 30 mg/kg LBH589, tumor reductionwas less evident and toxicity (skin dehydration and diarrhea)appeared; therefore, the experiments were focused on thedosage of 20 mg/kg.

Notably, acetylation of tubulin (Fig. 5, panel b) paralleledthe reduced growth in treated animals (correlation: r ¼�0.44, p < 0.01). Acetylation of histones was also observed,but no correlation with volumes at sacrifice was evident (datanot shown).

All xenografts were nodular; although no infiltration wasmacroscopically evident, tumors resulted in highly adherenceto surrounding tissues. Microscopically (H&E staining),tumors of the control group showed a high degree of necrosisand mitotic activity as typical in ATC. Overall, with the limitof the morphologic characteristics of ATC, the pathologistdescribed an evident decrease of necrosis in 20 mg/kg treatedanimals (data not shown). In 20 mg/kg treated animals, weobserved a significantly reduction of Ki67 (Fig. 5, panel c);notably, a positive correlation was observed between volumesat sacrifice and ki67 (r ¼ 0.48, p < 0.05) and between ki67and acetyl-tubulin (r ¼ 0.48, p < 0.05).

TUNEL analysis of in vivo tumors was performed. Apo-ptotic nuclei were double in tumors of 20 mg/kg LBH589-treated animals versus controls (six apoptotic cells/field intreated tumors as compared to three apoptotic cells/field inuntreated tumors). The results paralleled in vitro data; how-ever, the high level of necrosis, typical of ATC, stronglyreduces the number of fields where apoptosis can be eval-uated and makes the results less evident. As far as side effectsare concerned, only 30 mg/kg group animals presented skindehydration and diarrhea. At sacrifice, body weight wasslightly decreased in treated animals (10 mg/kg: �5%; 20 mg/kg: �8%, 30 mg/kg: �13%). No histological alterationsoccurred in any of the organs we analyzed.

DiscussionIn this study, we demonstrated that the DCI LBH589 is apotent cytotoxic drug in ATC both in vitro and in vivo.LBH589 is much more effective than VPA, the other DCI wetested in the same cell lines, and than other DCIs previouslyused against undifferentiated thyroid cancer cell, which areeffective only in the micromolar to millimolar range.6,8,10,12,19

The anticancer activity of LBH589 observed here is in linewith our recent report on breast cancer20 and with previousstudies from other authors in pancreatic,21 lung22 and biliarytract23 cancers as well as in hematological malignancieslike T-cell lymphoma,24 multiple myeloma25 and refractoryleukaemia.26,27

The mechanisms underlying the cytotoxic effect ofLBH589 on ATC cell lines included both apoptosis-inductionand cell cycle arrest. Apoptosis was demonstrated by theincreased percentage of cells in sub-G1, by the formation ofcytoplasmatic nucleosomes and by the activation of caspase3. LBH589 effect on cell cycle progression showed a distinc-tive feature: in fact, G1 arrest was already evident when cellswere treated with LBH589 at lower doses (5–25 nM), whereasthe arrest in G2/M appeared after treatment with higherdoses of the drug (�25 nM). This behavior is in agreementwith previous observations showing that DCI usually deter-mine cytotoxicity at higher doses and induce G1 arrest atlower doses.28,29

The primary molecular mechanism of DCI action is to al-ter the acetylation status of the core histone proteins, thusfacilitating chromatin remodeling with consequent alterationin gene expression,30 and cell differentiation.7 In agreementwith this, we showed that LBH589 acetylates histones of ana-plastic thyroid cancer cells, finally leading to the upregulationof p21 and the downregulation of cyclin D1. However, evenif histones were considered the canonical substrate of acetyla-tion, several studies have challenged this minimalist paradigmand implicated protein acetylation in a surprisingly diversearray of cellular processes including protein traffic, apoptosisand cell motility.31,32 Here, we demonstrated for the firsttime that low doses of LBH589 lead to microtubule stabiliza-tion as evidenced by increased tubulin acetylation and bundleformation. Tubulin acetylation is an established marker ofmicrotubule stability33–35; the suppression of spindle-microtu-bule dynamics slow or block mitosis at the metaphase-ana-phase transition, thus facilitating apoptotic cell death.Accordingly, LBH589 treatment resulted in a block duringmitosis as evidenced by the presence of multiple nuclei. Wepropose that LBH589 cytotoxic effects in anaplastic thyroidcancer cells can be mediated through microtubule stabiliza-tion. The effects on cell cycle progression are evident at 5–25nM, whereas 50–100 nM LBH589 is necessary to modulatecell cycle gene expression, suggesting that the transcriptionaleffects of LBH589 require higher doses than those necessaryto affect the nonhistonic proteins.

The effects of LBH589 are attained at concentrationsachievable in humans. In fact, in a Phase I study on intrave-nous LBH589, Cmax reached up to 200 nM14 and preliminaryresults36 from an ongoing Phase I pharmacokinetic study onoral LBH589, conducted in patients with solid tumors andhematologic malignancies, a steady state Cmax ranged from15 to 35 nM.

On basis of the in vitro data, we performed in vivo studiesusing the SCID xenograft model implanted with CAL-62cells. Animal data confirmed the cytotoxic properties ofLBH589, showing that the drug significantly impaired tumorgrowth. The maximum effect (final tumor volume 2.5-foldsmaller than in untreated animals) was evident at the dose of20 mg/kg; at this dose no relevant side effects were observed.In tumors of treated animals, a significant reduction of Ki67

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and a decrease of necrosis were observed, and acetylation oftubulin paralleled the reduced growth. The negative correla-tion between acetyl-tubulin and volumes at sacrifice andbetween acetyl-tubulin and ki67 reinforced the opinion thatthe levels of tubulin acetylation have a role in the inhibitionof tumor growth.

In conclusion, the anticancer activity of LBH589 opens uptherapeutic perspectives for the anaplastic thyroid tumor,which do not respond to conventional therapy. Translationaland clinical studies will ultimately determine the clinical util-

ity and safety of LBH589, used alone or in combination withchemotherapics, as an option for the treatment of this kindof tumor.

AcknowledgementsWe thank Prof. G. Bussolati for his critical comments and advice byreading the manuscript. We thank Novartis Pharma AG, Basel, Switzerlandfor providing us with LBH589. This study was supported by the Project‘‘Alfieri 2007’’, Fondazione CRT, Turin, by MIUR and by Regione Piemonteto G.B.

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Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo

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